With the increasing use of methotrexate (MTX) to treat arthritis, liver toxicity, which limits use of this drug, is of increasing concern. Because dihydrofolate reductase (DHFR) is the specific site of inhibition of this folic acid antagonist, the function of DHFR in liver, particularly human liver, is an important research objective. We are continuing our studies to isolate and characterize hepatic DHFR from human liver samples as well as from other species. We have found differences in human liver DHFR activity depending upon the tissue source. Not surprisingly, DHFR activity could not be detected in preparations of human liver from autopsies. Human liver samples obtained during surgery, on the other hand, gave measurable levels of DHFR activity when homogenized with appropriate proteolytic enzyme inhibitors. The DHFR levels of the human liver samples tested to date are quite low compared to preparations of chicken, beef, and sheep liver, i.e. 1/100 the level of activity in beef. Human hepatic DHFR, partially purified by affinity chromatography on MTX-sepharose followed by isoelectric focusing, has a mol. wt. of 22,000 and an isoelectric point of 6.8-7.0. Continuation of the studies to obtain more detailed information on carotenoid metabolism in normal humans: HPLC analysis of human plasma gave a peak of some 10-15% of total carotenoids different from any known standard compounds. Current studies suggest that the unknown compound may be hydroxy-alpha-carotene.